Purification of recombinant proteins and peptides for pharmaceutical use are mainly performed using liquid chromatographic methods. Chromatographic methods are characterised by their stationary phase attributes, e.g. reversed phase chromatography for hydrophobic chromatographic resins, however, a number of parameters affect separation such as the mobile phase composition. Ion-exchange chromatography (IEC) is characterised by a charged surface of the stationary phase and the use of buffer, salt and control of pH for the mobile phase composition. IEC has proven very efficient in separation of both related and unrelated impurities, however, some impurities may still be very difficult to remove, e.g. if the same charge of target polypeptide and impurity is present.
GB 694530 and GB 729670 describe the crystallisation of insulin in the presence of quinoline or acridine-like substances and of phenolic nature, respectively, also in the presence of zinc.
U.S. Pat. No. 5,504,188 describes the preparation of insulin analogue crystals (LysPro) at pH 5.5-6.5 in the presence of (among others) zinc ions.
WO 98/34953 describes the crystallisation of a protein with lysine side-chain carrying a lipophilic substituent (insulin detemir) in a solution containing zinc ions, crystallisation being accomplished by adjusting solution pH from acidic to pH 7-10.
U.S. Pat. No. 3,907,676 discloses a process of reducing the antigenicity of insulin recovered from pancreas glands of domestic mammals, particularly pork and bovine pancreas glands, and containing antigenic insulin-like substances with a molecular weight about 6,000 together with some antigenic proteins of pancreatic origin with a molecular weight above 6,000. The reduction in antigenicity is obtained by subjecting the insulin to column chromatography on an anion exchanger which is preferably strongly basic while using a water-containing monohydric aliphatic alcohol as eluent, and collecting the eluate fractions containing insulin free or essentially free of the impurities referred to.
EP 1 071 703 disclose that addition of calcium ions to the mobile phase have shown to provide an improved selectivity between glycosylated and non-glycosylated species (specifically for insulin) compared to conventional RPC methods (employing monovalent ions).
U.S. Pat. No. 3,649,456 basically describes the buffer exchange of a polypeptide on a RPC-like stationary phase from an aqueous solution to organic solvent containing solution in the presence of various salts including calcium chloride.
U.S. Pat. No. 5,633,350 describes the separation of K-vitamin dependent proteins from non-K-vitamin dependent proteins in the presence of calcium ions on ion-exchange resins.
GB 2173503A describes a process for the purification of insulin using a weakly acid cation exchanger preferably with a hydrophobic matrix is provided. Fractionation of the insulin is obtained by step-wise or continuous alteration of the concentration of the solvent in the acid pH range.
U.S. Pat. No. 4,129,560 describes a process for the purification of high molecular weight peptides, which have a tendency to associate, by ion exchanger chromatography in aqueous buffered solvents on acid or basic ion exchangers, which comprises dissolving non ionic detergents in the buffered solvents.
US 2005-080000A discloses a method for the chromatographic purification of preproinsulins, in which higher molecular weight substances are removed from an aqueous solution of preproinsulin by a first chromatography on an anion exchanger in flow-through mode and a subsequent second chromatography on a cation exchanger in adsorption mode, and to a method for preparing insulins, which includes the method for preparing preproinsulins.
US 2006-167221A discloses the extraction and isolation of insulins from recombinant sources, particularly those expressed in and secreted by yeasts. Organic solvents have been used to extract host surface bound forms of insulin peptide. In addition, procedures for combining the steps medium clarification, solvent extraction and chromatography, in order to effect the simultaneous isolation and purification of soluble and membrane bound forms of insulin, is disclosed.
U.S. Pat. No. 5,278,284 describes a method of removing a valuable protein from a complex solution and recovering the valuable protein in purified form consists of adding a silica gel sorbant having a pore size approximately the molecular size of the protein to a solution containing the protein, allowing the protein to be sorbed onto the sorbant, separating the sorbant from the solution and then recovering the protein from the sorbant.
U.S. Pat. No. 6,451,987 discloses an ion exchange chromatography process for purifying a peptide from a mixture containing the peptide and related impurities, and to an industrial method including such ion exchange chromatography process.
Some (often related) impurities are often difficult to remove or reduce due to the peak shape, e.g. an impurity eluting in the peak fronting or in the trailing edge. Thus, insufficient purity of a protein of interest may result.